High-Throughput Analysis Reveals miRNA Upregulating α-2,6-Sialic Acid through Direct miRNA–mRNA Interactions

Chemical biology has revealed the importance of sialic acids as a major signal in physiology and disease. The terminal modification α-2,6-sialic acid is controlled by the enzymes ST6GAL1 and ST6GAL2. Dysregulation of this glycan impacts immunological recognition and cancer development. microRNAs (miRNA, miR), noncoding RNAs that downregulate protein expression, are important regulators of glycosylation. Using our recently developed high-throughput fluorescence assay (miRFluR), we comprehensively mapped the miRNA regulatory landscape of α-2,6-sialyltransferases ST6GAL1 and ST6GAL2. We found, contrary to expectations, the majority of miRNAs upregulate ST6GAL1 and α-2,6-sialylation in a variety of cancer cells. In contrast, miRNAs that regulate ST6GAL2 were predominantly downregulatory. Mutational analysis identified direct binding sites in the 3′-untranslated region (UTR) responsible for upregulation, confirming it is a direct effect. The miRNA binding proteins AGO2 and FXR1 were required for upregulation. Our results upend common assumptions surrounding miRNA, arguing that upregulation by these noncoding RNA is common. Indeed, for some proteins, upregulation may be the dominant function of miRNA. Our work also suggests that upregulatory miRNAs enhance overexpression of ST6GAL1 and α-2,6-sialylation, providing another potential pathway to explain the dysregulation observed in cancer and other disease states.

obtained data for 2,161 miRNAs for ST6GAL1 and 2,166 miRNAs for ST6GAL2 out of 2601 total miRNAs screened. The Cer/mCh ratio for each miRNA was then normalized to the Cer/mCh ratio for the NTC within that plate and error was propagated. Data from all plates were then combined and z-scores calculated. A z-score of ±1.965, corresponding to a two-tailed pvalue of 0.05, was used as a threshold for significance. Post-analysis we identified 69 miRNA hits for ST6GAL1 and 62 for ST6GAL2 (see Fig. 1, Supplementary Fig. 3 and Supplementary Data 1).

RT-qPCR:
Total RNA was isolated from cells treated as in Western blot experiments using TRIzol reagent (catalog #: 15596018, Invitrogen) according to the manufacturer's instructions. RNA concentrations were measured using NanoDrop, and high-quality isolated total RNA was reverse-transcribed to cDNA using Superscript III Cells Direct cDNA synthesis kit (catalog #: 18080300, Invitrogen). Reverse transcription quantitative PCR (RT-qPCR) was performed using the SYBR Green method and cycle threshold values (Ct) were obtained using an Applied Biosystem (ABI) 7500 Real-Time PCR machine and normalized to housekeeping gene GAPDH. The primer sequences used in RT-qPCR can be found in Table S1. All analysis was done in biological triplicate

SNA Staining Assay:
Cells were seeded onto sterile 22 × 22 no. 1 coverslips placed into 35 mm dishes at a density of 5 × 10 4 cells/ml in standard media. After 24h, cells were transfected with miRNA mimics or antimiRs as in the Western blot section. At 48 h post-transfection, cells were washed with PBS (3×, 2 mL) and fixed with 4 % paraformaldehyde for 15 min. Cells were again washed with PBS (3×, 2 mL). blocked using 10 % BSA in PBS for 1h in incubator (37 ℃, 5% CO2) and Cy3-SNA was added (1:300 in 10 mM HEPES, 0.15 M NaCl, 0.1 mM CaCl2, pH 7.5, Vector Laboratories, catalog # CL-1303). After 1 h in the incubator, coverslips were washed (PBS, 3×), and cells were counterstained with Hoechst 33342 (1 µg/mL in PBS, 15 min in incubator). The coverslips were then mounted onto slides with 60 µl of mounting media (90% glycerol in PBS) and imaged with a Zeiss fluorescent microscope (Camera: Axiocam 305 mono, software: ZEN 3.2 pro). For each biological replicate, 5 fields were obtained. Specificity of SNA staining was confirmed by using neuraminidase (gift from Dr. Matthew Macauley) prior to SNA staining. All analysis was done in biological triplicate. For data analysis, the ZOI method in the ZEN 3.2 pro software was used to quantify the fluorescence signal in membrane of all cells. Signal was normalized to cell count using the Hoechst staining to count nuclei in the software. Final data was normalized to the NTC for each biological replicate. A paired t-test was used to compare NTC with miRNA or anti-miR.

S5
The 3'-UTR sequence of ST6GAL1 and the three miRNA sequences (miR-221-5p, miR-212-5p, miR-4531) were analyzed with the RNAhybrid tool which calculates a minimal free energy hybridization of target RNA sequence and miRNA. The two stable predicted miRNA: mRNA interaction sites were selected for designing mutant pFmiR-sensors. Multiple mutation sites were designed and mutant sequences were ordered for synthesis from GenScript Biotech or Integrated DNA Technologies (IDT). Each synthesized mutant fragment (221-MUTA-gBlock, 221-MUTB-gBlock, 212-MUTA-gBlock, 212-MUTB-gBlock) was amplified by standard PCR machine (Bio-Rad), using the primer sequences found in Table S1. Amplicons were cleaned up using Monarch PCR & DNA cleanup kit (catalog #: T1030S, NEB). The NucleoSpin Gel and PCR Clean-up XS kit (REF. #: 740611.50) was used for DNA gel extraction when needed to exclude non-specific bands. The amplicons were ligated into the empty pFmiR plasmid after enzymatic digestion using a pair of restriction enzymes for each gBlock (221-MUTA: NheI, PasI; 221-MUTB: PasI, BamHI; 212-MUTA: NheI, PasI; 212-MUTB: SwaI, BamHI). Sequences for the mutant pFmiR-ST6GAL1 sensors were then verified by sequencing and used in the miRFluR assay as described previously. A minimum of 3-wells were transfected per sensor and the analysis was done in 2 independent experiments.

Multi-Site Mutagenesis: ST6GAL2:
The 3'-UTR sequence of ST6GAL2 and the two miRNA sequences (miR-3619-5p, miR-30c-2-3p) were analyzed with the RNAhybrid tool which calculates a minimal free energy hybridization of target RNA sequence and miRNA. The two stable predicted miRNA: mRNA interaction sites were selected for designing mutant pFmiR-sensors. Multiple mutation sites were designed and mutant sequences were ordered for synthesis from GenScript Biotech or Integrated DNA Technologies (IDT). Each synthesized mutant fragment (3619-MUTA-gBlock, 3619-MUTB-gBlock, 30c-MUT-gBlock) was amplified by standard PCR machine (Bio-Rad), using the primer sequences found in Table S1. Amplicons were cleaned up using Monarch PCR & DNA cleanup kit (catalog #: T1030S, NEB). The NucleoSpin Gel and PCR Clean-up XS kit (REF. #: 740611.50) was used for DNA gel extraction when needed to exclude non-specific bands. The amplicons were ligated into the empty pFmiR plasmid after enzymatic digestion using a pair of restriction enzymes for each gBlock (3619-MUTA: AjuI, PasI; 3619-MUTB: Psil-v2, PasI; 30c-MUT: AjuI, PasI). Sequences for the mutant pFmiR-ST6GAL2 sensors were then verified by sequencing and used in the miRFluR assay as described previously. A minimum of 3wells were transfected per sensor and the analysis was done in 2 independent experiments.
Figure S12 | Predicted miRNA binding site analysis for α-2,6-sialyltransferases. a, Map of the binding sites for miRNA identified as hits by our miRFluR assay. Sites shown are the most stable hybridization sites predicted by RNAhybrid 19 . Annotations are given for every 300 bp. b, Map of the binding sites for miRNA identified as hits for ST6GAL2 by our miRFluR assay. Sites shown are the most stable hybridization sites predicted by RNAhybrid 19 . Annotations are given for every 600 bp. c, Bar graphs representing the percentage of AU content of four different predicted miR sites (canonical seed: perfect seed match, supplementary pairing: base pairing near to canonical site, non-canonical seed: imperfect seed match, compensatory pairing: base pairing near to noncanonical site) for all predicted up-miRs sites for ST6GAL1 and ST6GAL2. Sites considered in b are the most stable predicted sites based on RNAhybrid shown in a. d, Pie charts representing the distribution of miRNA site overlap within the 3'-UTR for ST6GAL1 and ST6GAL2. Sites are defined as overlapping if the annotated hybridization sites share nucleotides. Periwinkle blue: up-miRs with no overlap, turquoise blue: overlap between 2 or more up-miRs, green: overlap between a set of up-miRs and down-miRs, magenta: overlap between 2 or more down-miRs, red: down-miRs with no overlap. Percent is function of sum of miRNA hits. e and f, Alignment of down-miRs (ST6GAL1: 4531, ST6GAL2: 30c-2-3p) with predicted 3'UTR sites and their corresponding mutants. g, Bar graph of data from mutant miRFluR sensors as in d and e. Data was normalized to NTC in each sensor. For statistics data was compared to wildtype (WT) for each miRNA. All experiments were performed in biological triplicate. Errors are standard deviations. Standard t-test was used (* p < 0.05, ** < 0.01, *** <0.001).   Figure 5b. Arrows indicating the validated ST6GAL1 signal. g, RT-qPCR analysis for samples as in Figure 5g. All samples are normalized to GAPDH and NTC. All experiments were performed in biological triplicate. Errors are standard deviations. Standard t-test was used (* p < 0.05, ** < 0.01, *** <0.001).